The abuse of illegal substances with reinforcing addictive properties represents a major public health problem worldwide. For instance, in the United States of America, nearly 48 million people have been exposed to illegal drugs over a one-year period (Neurobiological Adaptations to Psychostimulants and opiates as basis of treatment development. In: New Medications for Drug Abuse, K. Severino, A. Olivito and T. Kosten, 2000). Thus, this health problem has serious and progressive deleterious effects on social, economic and medical areas in affected countries. Epidemiologically, the pyschostimulants such as cocaine and amphetamines, and to a lesser extent, opiate substances, like heroin and morphine, represent the most prevalent drugs causing the highest addictive morbidity worldwide. In developing countries like Mexico, the epidemiological data from the latest National Survey of Addictions (M. E. Medina-Mora and E. Rojas Guiot, Salud Mental, 26(2): 1-11, 2003) reported an alarming increase in the drug-intake of such substances in the central part of the country as well in cities located between Mexico and US border. At the clinical level, there are several co-morbid pathologies related to the addictive abuse of illegal substances, which fall into different categories. Firstly, the high death index related to the toxic effects induced by the overdose of such substances. Secondly, the induction of teratogenic effects in the newborn, which are frequently associated to the chronic abuse of illegal substances by addicted pregnant mothers. Finally, the high incidence of co-morbid diseases of acquiring viral infections such as the human immunodeficiency virus (HIV), frequently detected in heroin abusers, as well as the increased rates of crimes, violence and delinquency frequently associated to the drug-trade and drug-intake of such illegal substances. Thus, at the therapeutical level, there exist an urgent need to refocuse and establish straightforward goverment strategies, health programs and novel medications to fight efficiently against drug abuse to illegal substances.
The neurobiology of drug addiction began more than three decades ago and most of investigations have dealt with drugs' pharmacokinetics and pharmacodymamics. At the pharmacokinetic level, illegal substances of abuse such as cocaine, morphine and heroin, exhibit potent drug-reinforcing properties and specific pharmacokinetic profiles, which ultimately lead to their high addictive drug effects in the brain. Morphine is an alcaloid with a phenantrenic chemical structure (see example 1) obtained from the milky extract (opium gum) of the Papaver somniferum, and represents the main compound extracted (≧10%) together with other structurally-related compounds such as codeine, tebaine, and papaverine (C. P. O'Brien, Drug Abuse, In: The Pharmacological Basis of Therapeutics. Pp. 621-642, 10th ed. J. G. Hardman and L. E. Limbird, eds. McGraw Hill, New York, 2001). Morphine possesses a hydroxyl group in the third position and an alcoholic hydroxyl group in the sixth position placed within the phenolic ring structure. Conversely, heroin, a semi-synthetic derivative of the morphine, has two acetyl groups condensed in the aforementioned positions within the opiate phenantrenic ring structure (C. P. O'Brien, Drug Abuse, In: The Pharmacological Basis of Therapeutics. pp. 621.642, 10th ed. J. G. Hardman and L. E. Limbird, eds. McGraw Hill, New York, 2001). Both morphine and heroin are absorbed from the gastrointestinal and respiratory tract, including oral mucosa, as well as from the subcutaneous, intramuscular, intravascular and intrathecal spaces. These two opiate compounds display a striking similar pharmacokinetic profile, based on their high blood-brain barrier permeation capability, mostly due to their high lipophilic properties (C. P. O'Brien, Drug Abuse, In: The Pharmacological Basis of Therapeutics. pp. 621-642, 10th ed. J. G. Hardman and L. E. Limbird, eds. McGraw Hill, New York, 2001). In fact, heroin is relatively more lipophilic than morphine and thus permeates faster the blood-brain barrier than morphine. The main catabolic route of morphine mainly occurs in the liver and depends on enzymatic-dependent conjugation with glucuronic acid at both the three and six hydroxyl groups placed at the phenantrenic ring structure, producing endogenous metabolites compounds, such as morphine 3-, morphine 6-, and to a lesser extent, morphine 3-6-glucuronide. These catabolic intermediate compounds, represent the structural secretory and/or excretory forms of morphine in the urine. Moreover, morphine-6-glucuronide has been shown to display a potent analgesic and psychotropic, drug-reinforcing effects in the brain. Thus, morphine metabolites generated from the liver into the bloodstream, rapidly permeate the blood-brain barrier and activate the mu opioid receptor subtype in the brain reward pathways mediating the reinforcing effects of drug of abuse (L. M. Kamendulis et al., J. Pharmacol. Exper. Ther. 279:713-717, 1996; C. W. Hutto Jr. y W. Crowder, Pharmacol. Biochem. Behav. 58(1):133-140, 1997; A. J. Halliday et al., Life Sci. 65(2)225-236, 1999 and D. E. Selley et al., Biochem. Pharmacol. 62:447-455, 2001). In fact, recent pharmacokinetic studies (see review and references therein in J. Halliday et al., Life Sci. 65(2)225-236, 1999) support the idea that the analgesic and/or addictive actions of morphine in CNS are not directly and predominantly mediated by morphine itself, but largely exerted by its glucuronated active metabolites such as the morphine-6-glucuronide. So far, several studies (L. M. Kamendulis et al., J. Pharmacol. Exper. Ther. 279:713-717, 1996 and A. J. Halliday et al., Life Sci. 65(2)225-236, 1999) have shown similar pharmacokinetic and pharmacodynamic mechanisms for heroin. Thus, once heroin is administered, a large fraction of the drug is rapidly catabolized in the plasma and/or liver into 6-monoacetyl-morphine, and subsequently catabolized into morphine and finally converted into morphine-6-glucuronide, before reaching their neuronal targets (e.g., mu opioid receptor) (R. E. Aderjan and G. Skopp, Ther. Drug Monit., 20(5): 561-9, 1998). These findings support the current concept that the pharmacological agonism of heroin and its endogenous metabolites (e.g., 6-monoacetyl-morphine and morphine) on the mu opioid receptor including the final biotransformation active metabolites (e.g., morphine-6-glucuronide) represents the pharmacodymamic mechanism by which these substances enhance their reinforcing addictive actions in the brain (a. J. Halliday et al., Life Sci. 65(2): 225-236, 1999, D. E. Selley et al., Biochem. Pharmacol. 62:447-455, 2001 and C. P. O'Brien, Drug Abuse, In: The Pharmacological Basis of Therapeutics. pp. 621-642, 10th ed. J. G. Hardman and L. E. Limbird, eds. McGraw Hill, New York, 2001).
Several pharmacodymic studies (see reviewed works in E. J. Nestler, Nat. Neuroci. 5:1076-1079, 2002 and P. N. Deslandes et al., J. Pharmacy and Pharmacol. 54:885-895, 2002) have shown that chronic abuse to both heroin and morphine leads to the development and establishment of specific long-term changes at the cellular and molecular level that ultimetaly produces the expression of biological neuroadaptations to opiate addiction. Moreover, these neuronal changes produce important electrophysiological, neurochemical and genomic changes, which are progressively established and consolidated upon a long-term period (e.g., years) in the brain during drug addiction. Therefore, the behavioral changes occurring during opiate addiction to these substance of abuse in the individual, follow a time-course of increased complexity and intensity with regard to the drug addiction symptomology. For instance, the repetitive administration of heroin by an addict, produces an increase stereotyped compulsive behaviors leading to uncontrolled drug-intake behaviors, associated with stereotyped rites of administration, initially accompanied by pharmacological tolerance and subsequently by physical signs and symptoms of drug withdrawal after acute suppression of the opiate drug (K. Severino et al., Ann. NY Acad. Sci 909: 51-87, 2000). Thus, heroin-intake behavior becomes the highest priority and necessity in the addicted individual, leading to the reinstatement of compulsive drug-intake and drug-seeking behaviors normally observed during drug withdrawal or abstinence. The neuroadaptative changes occurring during opiate addiction is primarily caused by the pharmacological actions displayed by the repetitive exposure of the drug over a clustered group of neurons localized in different areas of the brain (K. Severino et al., Ann. NY Acad. Sci 909: 51-87, 2000). These brain areas include the locus coeruleus, hippocampus, lateral hypothalamus, ventral-tegmental area, amygdaloid complex, nucleus accumbens and prefrontal cortex, which structurally comprised the neuroanatomical substrate and neural pathways where opiate substances and other illegal drugs of abuse (e.g., cocaine) mainly exert their drug-rewarding and drug-reinforcing activities (K. Severino et al., Ann. NY Acad. Sci 909: 51-87, 2000). In this context, the chronic administration of both morphine and heroin induces the development of a series of homeostatic cellular and molecular adaptative responses on neurons within the aforementioned brain structures impinged by the drug. Such adaptative responses involve several electrophysiological, biochemical and genomic alterations seen during drug addiction, which altogether, are produced to maintain and restore the pre-existing functional homeostasis of the implicated neural circuits and their operant neurons altered during drug abuse, prior to the compulsive drug-intake behavior (K. Severino et al., Ann. NY Acad. Sci 909: 51-87, 2000). Once these neuroadaptations have been established, the abrupt suspension of the drug-intake behavior enhances the development of new series of neurobiological changes and cellular adaptations in the neurons impinged by the drug, leading to the neuropathological basis that underlies the withdrawal syndrome during drug addiction. The withdrawal syndrome produced by both morphine and heroin in the addicted individual, as opposed to the withdrawal syndrome induced by cocaine and amphetamine, is characterized by highly intense physical and psychological alterations in the addicted individual (H. Ghodse, Drugs of abuse and dependence. In: Drugs and Addictive Behavior, a guide to treatment, Blackwell Science Ltd, ed., Oxford, UK, pp. 72-119, 1995; G. F. Koob, Ann. N.Y.: Acad. Sci. Vol. 909:185 2000 and K. Severino et al., Ann. N.Y. Acad. Sci 909: 51-87, 2000). Clinically, the withdrawal syndrome is characterized by four different stages developed in a progressive or gradual time-course. During the first 1-7 hours, the addict under abstinence develops behavioral manifestations characterized by a compulsive craving and extreme anxiety for drug-intake. During a second stage (after 8-15 hours), physical alterations such as intense lacrimation, extreme sweating, rinorrhea and lethargy are added to the initial drug symptomology. Further on, after 16-24 hours upon continuing drug withdrawal, physical signs such as mydriasis, piloerection, muscular cramps and changes in body temperature (e.g., intense cold and heat perception) in addition to diffuse algias, anorexia and irritability may appear as well. Subsequently, upon pesistent withdrawal (e.g., 2-6 days), other physical and behavioral signs may appear which include insomnia, fever, motor delay, abdominal pain, vomiting and diarrhea as well as increased abnormal breathing, including changes in pulse frequency and blood pressure. Thus, from the perspective of symptomology occurring in drug addiction, the duration and severity of morphine and heroin withdrawal depends on several pharmacokinetic and pharmacodynamic factors. Moreover, there has been reported that the severity of opiate withdrawal syndrome depends on several pharmacological and biological aspects, which include the daily amount of drug-intake (e.g., dose injected by the individual), the period of time of drug use and/or abuse, in addition to the physical and personality status of the individual affecting drug-intake response.
Thus, given the complexity of the natural history of the morphine/heroin addictive pathology, few currently available pharmacological treatments have been designed to modify the pharmacodynamic mechanisms by which these opiate substances produce their drug-reinforcing actions once they bind their specific receptor sites at their targeted neurons (D. M. Grilly, Opioids (narcotics) and their antagonists. In: Drugs and human behavior, 4th ed. pp. 238-262, Allyn and Bacon, eds. USA, 2002). In this context, the acute opiate detoxification treatment represents the initial and most currently used pharmacotherapeutic approach to treat clinically chronic addicts, which becomes a medical priority and emergency to relieve the individual's physical signs and symptoms of drug withdrawal, which are commonly associated with physiological, endocrinological and chemical disturbances induced by drug addiction. For example, mu opioid receptor partial agonists such as methadone and buprenorphine, in combination with benzodiazepines and/or sedative neuroleptics are commonly prescribed and administered for the acute desintoxication treatment to opiates. As opposed to the acute detoxification procedures used to treat opiate addiction, the substitution therapy using opiate substances such as methadone and/or buprenorphine as well as opioid receptor antagonists, such as naloxone and/or naltrexone, are not entirely recommended during opiate withdrawal, because they exacerbate the demand of drug-intake behavior of the parent opiate compounds that elicited or installed the former drug addictive state in the individual. Under normal circumstances, the treatment and maintenance of opiate withdrawal syndrome requires hospitalization and clinical care with the support of specialized medical personnel, which commonly results to be highly expensive. Likewise to the withdrawal syndrome, the complete morphine and/or heroin detoxification (supression of drug-intake behavior) in addicted individuals is an important health issue to be pursued. Based on the wide range of abnormal functional changes established after a long-term period in the brain produced by chronic opiate abuse, it is easy to understand the difficulties to re-establish the homeostatic function of the brain, prior to drug-intake, by the current available detoxification therapies. Thus, despite these therapeutic limitations, an ideal detoxification treatment must be address to meet specific medical criteria described as follows. Firstly, it should be directed to block or blunt the physiological and psychological opiate dependence in order to re-establish the homeostatic balance of those neural systems chronically dysregulated by opiate substances. Secondly, the detoxification treatments should inhibit those pertinent physical and behavioral changes that appear to be exacerbated during drug withdrawal induced by therapeutic interventions, thereby resulting in a tolerable experience and safety treatments. Additionally, it should provide a complete suspension of the individual's drug-intake behavior, thus reorientating the addicted individuals to other alternate available non-pharmacological treatments (e.g., psychotherapy and counseling). Thereafter, once the complete opiate detoxification therapy is reached, the final medical goal to be approached is the prevention of subsequent relapses to opiate abuse. Thus, from a general medical viewpoint, the therapeutic challenges to blunt morphine/heroin addiction are enormous and, in most of cases, difficult to improve. The main obstacles faced by both pharmacological and non pharmacological-based treatments, are the lack of an adequate number of specialized clinics or hospitals, the high economical costs of therapy usually billed to the patient and, most importantly, the absence of either patient follow-up programs (i.e., years) or continuos clinical evaluation as wella as the lack of application of long-term psychotherapy support to prevent drug-relapse. In addition, another major problem facing most of the current anti-addictive treatments against opiate abuse is the side-effect toxicity resulting from long-term dosification of single or combined pharmacological agents (K. Severino et al., Ann. NY Acad. Sci 909: 51-87, 2000). For example, methadone and buprenorphine, two long-lasting partial agonist of the mu opioid receptor, represent the most common substitution therapeutic drugs used today to blunt opiate withdrawal syndrome or to prolong opiate abstinence (M. J. Kreek, Ann. N.Y. Acad. Sci, 909: 186-216, 2000). In addition, α2 adrenergic receptor agonists such as clonidine, guanfacine and/or lofexidine represent another set of compounds used quite frequently in detoxification therapies to amielorate withdrawal signs and symptoms caused by the abrupt supression of opiate drugs (M. J. Kreek, Ann. N.Y. Acad. Sci, 909:186-216, 2000). However, besides of their widely use in long-term detoxification therapies and/or treatment maintenance of abstinence, these drugs have been shown to induce several toxic side-effects. For example, methadone, buprenorphine and pentazocine have been reported to produce sleep disorders, anxiety and severe cognitive and emotional impairment. Additionally, α2 adrenergic receptor agonists have been reported to produce sedation, hypotension, extreme anxiety and asthenia upon long-term administration. In addition, patients receiving opiate-substitution with methadone, may not surprisingly, show the development of signs and symptoms of opiate-dependence due that this mu opioid receptor partial agonist produces same neurochemical, cellular and molecular neuroadaptative changes in the brain, as those reported for both morphine and heroin during opiate addiction (M. J. Kreek, Ann. N.Y. Acad. Sci, 909:186-216, 2000). Other available drugs currently used to prolong abstinence and to relapse prevention against morphine/heroin addiction in detoxified patients comprise the mu opioid receptor antagonists, naloxone and naltrexone. The toxic side effects often seen during the long-term administration of these compounds are mostly due to the blockade of the endogenous opioid transmission systems in the brain, leading to impairement of both cognitive and emotional brain functions, among many other physiological activities (M. J. Kreek, Ann. N.Y. acad. Sci, 909:186-216, 2000).
Thus far, one major conclusion drawn from the above described pharmacological therapies currently used to approach detoxification against opiate abuse including long-term maintenance treatments for drug-withdrawal and relapse-prevention against morphine/heroin addiction, is that none of these pharmacological treatments have shown an optimum efficacy. This conclusion is based on the fact that these drugs produce important toxic side-effects in patients receiving long-term maintenance of abstinence and/or relapse-prevention (T. Kosten and D. Biegel, Expert Rev. Vaccines, 1(3): 89-97, 2002). Thus, there is an urgent need to develop and validate novel anti-addictive therapeutic strategies, based on the synthesis, application and validation of highly effective new drug formulations, displaying minimum toxicity and no detected side-effects, when pretend to be use in the long-term therapies for acute detoxification and long-term maintenance of morphine/heroin abstinence.
For this reason, here are given and shown all the reports and documents concerning the state of the art of the development and application of techniques related to the present invention, which are detailed herein and are also included to be used only as reference material.
In this context, different groups have designed, applied and validated alternative therapeutic strategies in experimental animal models, which share a common pharmacokinetic mechanism. Thus, conversely to the classical anti-addictive pharmacology, this latter mechanism is based on altering the drug's pharmacokinetics by decreasing significantly or blunting the blood-brain barrier permeation of the “free” unbound drug in plasma, which ultimately represents the fraction of drug in plasma that permeates the brain causing the high reinforcing and rewarding effects in the addicted individual. All of these experimental approaches have been focused to decrease significantly or prevent the blood-brain barrier permeation of drugs of abuse, by enhancing the binding of the “free” unbound fraction of drug in plasma by specific antibodies, which recognize and bind with high specificity and avidity to these drugs in the blood. As immunoglobulins (antibodies) do not normally permeate the blood-brain barrier, the plasma fraction of “free-unbound drug”, which is the available pool of drug that permeate the blood brain barrier, interact with immunoglobulins establishing drug-antibody complexes, which ultimaltely decreases significantly this fraction of free-unbound drug in plasma. This change in the drug's pharmacokinetics in plasma, leads to altered changes in drug's pharmacodynamics in the brain, thus blunting or abolishing the activity of addictive drugs on their specific targeted neurons. These latter pharmacodynamic changes ultimately lead to blunt both the development of the reinforcing activities and the rewarding pleasant effects induced by drugs of abuse in the brain. The main pharmacokinetic property shared by most drugs of abuse, is the high blood-brain permeation activity, which represents the basic and crucial mechanism, by which most of the potent drugs of abuse produced their highly intense reinforcing actions in the brain, leading to the continous drug-intake and drug-seeking behaviors display by individuals upon exposure to these chemical compounds. In this context, the generation of specific serum antibodies against drugs of abuse represents an alternate therapeutical approach to blunt or prevent the blood-brain barrier permeation of drugs of abuse from reaching its neuronal targets. This antibody antagonism approach preventing drug's permeation into the brain has been shown to enhance an immunoprotective effect against drug-intake and drug-seeking behaviors, as demonstrated for cocaine, nicotine, PCP and amphetamines in rodents (see an account of reviewed works and references therein in T. Kosten and D. Biegel, Expert Rev. Vaccines, 1(3): 89-97, 2002 and K. Kantak, Drugs, 63(4): 342-252, 2003). With regard to cocaine, several research groups were able to develop and apply different experimental strategies based on the design, synthesis, application and validation of several immunogenic preparations of carrier proteins with covalently haptenized cocaine (Kantak et al., Psychopharmacology 148:251-262, 2000; Fox, B. S. et al., Nat. Medicine, 2:1129-1132, 1996; Sparenborg et al., Therapeutics 293(3): 952-961, 2000; Carrera et al. Nature, 378:727-730, 1995, Carrera, R. et al., Proc. Nat. Acad. Sci, USA, 97(11)6202-6206, 2000). Some high molecular weight proteins such as BSA and KLH have been used as carriers to covalently link cocaine using standard chemical covalent coupling procedures. In this way, following active immunization protocols in rodents, some of these immunogens have shown capabilities to generate low to moderate antibody titer responses against this drug of abuse in actively vaccinated animals. Moreover, other experimental approaches conferring immunoprotective effects against cocaine addiction have been explored by enhancing the generation of conventional and/or catalytic monoclonal antibodies administered during passive immunization protocols against this psychoactive drug in rodents (Metz et al., Proc. Natl. Acad. Sci. USA 95:10176-10180, 1998; Fox et al., Nat. Med. 2:1129-1132, 1996 and Landry et al., Science, 239:1899-1901, 1993). The immunoprotective effects against cocaine addiction using these immunological-based experimental strategies have been explored by detecting the abolishment of the drug-reinforcing behaviors in rodents in combined pharmacological and operant-behavioral paradigms. These experimental strategies share a common anti-addictive mechanism, which relies in the significant reduction and/or complete inhibition of the blood-brain permeation of the “free” unbound fraction of cocaine in plasma. Thus, in actively vaccinated hyperimmune animals, the fraction of “free” unbound of drug in plasma is significantly reduced, once specific serum antibodies in the blood bind to the psychoactive drug (Kantak et al., Psychopharmacology, 148:251-262, 2000; Carrera et al., Proc. Natl. Acad. Sci. USA, 97(11):6202-6206; Carrera et al., Nature, 378:727-730, 1995). Alternatively, monoclonal antibodies raised against cocaine, may bind the “free” unbound fraction of cocaine after being passively transferred into the blood (Metz et al., Proc. Natl. Acad. Sci. USA 95:10176-10180, 1998; Fox et al., Nat. Med. 2:1129-1132, 1996, Benowitz, Pharmacol. Toxicol. 72:3-12, 1993). In both cases, the common immunological neutralizing mechanism, which promotes altered changes in cocaine pharmacokinetics, leads to the significant decrement or complete inhibition of drug's entry into the brain, thereby decreasing or blunting the targeting of cocaine to the specific neuronal membrane dopamine reuptake transporter. This latter antibody-mediated mechanism inducing altered changes in cocaine's pharmacodynamic in the brain, would lead to changes in the synaptic level of amine neurotransmitters, abolishing the evoked-dependent increase in the central catecholaminergic tone, normally seen after cocaine's entry into the brain in addictive individuals. The final behavioral scenario that results from these altered changes in cocaine pharmacokinetics and neurochemical events is the lack of the intensified rewarding effects induced by this potent reinforcing drug in the brain of mammals. Thus, once cocaine has been neutralized to produce its reinforcing and rewarding effects in hyperimmune animals, the reinforcing properties of this drug will be lost upon a subsequent drug exposure, as demonstrated by the supression of drug-seeking and drug-intake behaviors in such hyperimmune vaccinated animals (rodents) seen with use of some immunogenic conjugates of cocaine.
In summary, most of the aforementioned pre-clinical studies have shown the feasibility of using antibody-based antagonism approach for blunting drug-taking and drug-seeking behaviors in rodents. However, the type of the carriers proteins (e.g., BSA and KLH) used in the preparation of the immunogenic conjugates (vaccines) used in these studies preclude its potential use in vaccine formulations for use in human immunization protocols (Carrera et al., Proc. Natl. Acad. Sci. USA, 2001; Carrera et al., Proc. Natl. Acad. Sci. USA, 2000; Carrera et al., Nature, 378:727-730, 1995; Kantak et al., Psychopharmacology, 148:251-262, 2000; Ettinger et al., Pharmacol. Biochem. Behav. 58:215-220, 1997 and Fox, Drug and Alcohol Depend. 48:153-158, 1997). Furthermore, the synthesis of conventional andor catalytic mouse anti-cocaine monoclonal antibodies used as potential passive immunotherapy for addition in experimental animals (rodents), has the main limitation in conferring immunoprotection in a short-term period when passively administered. This is mostly due to the fast metabolic clearance of these murine immunoglobulins from serum of passively immunized animals other than mice (Goldsby et al., Vaccines, In: Kuby Immunology, 4th ed. Freeman and Co. New Cork, N.Y., pp. 449-466, 2000). Moreover, the potential use of the available murine anti-cocaine monoclonal antibodies as immunological therapeutic agents against cocaine addiction in humans, requires the use of DNA recombinant techniques, so as to “humanize” the Fc segment of murine immunoglobulins.
Finally, the potential application of this antibody-based antagonism against cocaine addiction in the human is a current issue under experimentation as a therapeutical approach. This immunopharmacological strategy was initially approached through the synthesis of an anti-cocaine vaccine formulation, structurally designed for human use, where cocaine was covalently conjugated to the recombinant β-subunit of the cholera toxin (used as carrier protein. At pre-clinical level, this conjugate showed moderate efficacies in triggering antibody responses in actively vaccinated rats that confered immunoprotective effects to prevent relapse to cocaine taking-behavior in this animal (Kantak et al., Psychopharmacology, 148:251-262, 2000). Additionally, active vaccination with this immunogen in human volunteers, used to test the safety and immunogenicity of this vaccine formulation, unfortunately, showed little promissory therapeutic effects, in this single Clinical Phase I study (T. Kosten et al., Vaccine 2559:1-9, 2001), due to the fact that this vaccine formulation showed a poor immunogenic capacity, producing low antibody titer responses [e.g., low concentration range (μg) of specific immunoglobulin/ml of serum] in most of the vaccinated subjects. In addition to the aforementioned experimental limitations, new anti-cocaine vaccines developed by different groups of research, are currently being under study, using different carrier proteins, in order to generate an improved immunogenicity against this psychoactive drug in both pre-clinical and Clinical Phase I studies. Once the immunogenic properties of these vaccine formulations are validated in humans in Clinical Phase I studies, it may become available for a subsequent evaluation in Clinical Phase II studies assessing the immunoprotecting capabilities of these vaccine formulations against cocaine addiction. For instance, it could be used Clinical Phase II studies by assessing the enhanced long-lasting humoral-based immunoprotection against cocaine addiction, in former drug addicts, exhibiting a prolong and controled abstinence but challenged to the pharmacological reacquisition of addictive cocaine-intake behavior.
In the case of tobacco addiction, at least two immunogenic preparations (vaccines) to the reinforcing psychoactive substance, namely nicotine, have been designed for human application (see an account of reviewed works and selected references therein in T. Kosten and D. Biegel, Expert Rev. Vaccines, 1(3): 89-97, 2002; K. Kantak, Drugs, 63(4): 341-352, 2003). Pre-clinical studies have demonstrated that these two vaccines were able to generate low to moderate serum titers of specific antibodies (i.e., 0.05-0.2 mg/ml of serum) against nicotine in actively vaccinated rodents. Moreover, active vaccination with these immunogenic preparations of nicotine, demonstrated to confer immunoprotection against the acquisition nicotine-intake behavior in intravenous drug-self-administration paradigms in rodents. The immunoprotective mechanism against nicotine addiction follows same pharmacokinetic mediated-mechanism described for cocaine addiction, that is, through the binding of the “free” unbound fraction of nicotine in plasma by specific serum antibodies, which prevents the blood-brain barrier permeation of this drug. Clinical Phase studies performed independently by Nabi Pharmaceuticals (Anti-nicotine vaccine NicVAX) and Xenova Pharmaceutical Group in Belgium, reported successful results on the evaluation of the toxic and immunogenic properties of these two vaccine formulations. The reports on the evaluation of the immunoprotection capabilities of these two vaccine formulations against nicotine addiction in former drug addicted volunteers in Clinical Phase II studies are expected to be ready in the next two coming years.
In fact, the development of experimental strategies focused in the design and synthesis of immunogenic preparations and the subsequent validation of vaccination protocols for treating specific forms of drug addiction, were pioneered approached for opiates such as morphine and heroin, but not for cocaine and nicotine addiction. Retrospectively, at the beginning of the 70s, different research groups showed the feasibility of raising a humoral immune response against these two opiate substances using vaccination protocols in experimental animal models, such as the rat and the rabbit (S. Spector and C. W. Parker, Science, 168:1347-1348, 1970; S. Spector, J. Pharmacol. Exp. Ther. 178:253-258, 1971; E. L. Adler and C. Liu, J. Immunol, 106:1684-1685, 1971; H. Van Vunakis et al., J. Pharmacol. Exp. Ther. 180:514-521, 1972; B. H. Wainer et al., Science, 176-1143-1145, 1972; B. H. Wainer et al., Science, 178:647-648, 1972 and B. H. Wainer et al., J. Immunol. 110:667-673, 1973). These experimental approaches were focused in generating polyclonal antibodies against morphine, displaying distinct immunological cross-recognition against heroin and structurally related opiate analogues (e.g., codeine, meperidine, and hydromorphone). These antibodies were generated for using in specific-designed immunoassays (i.e., radioimmunoassay and ELISA immunoenzymatic assays) to detect and measure morphine and related opiate substances in biological fluids from humans. These studies showed, for the first time, the succesfull achievement on the design and validation of the covalent condensation of the exposed free 3- and 6-hydroxyl groups in the phenantrenic ring of the morphine molecule to carrier proteins such as BSA, using standard organic chemistry procedures (procedures that are still used when approaching chemical synthesis of such immunogenic conjugates). In addition, it is worth to mention that none of these chemical procedures were never reported and claimed in patent registries and they are mostly considered as classical chemical procedures in textbooks of organic chemistry, when describing the covalent linkage of the free 3- and 6-hydroxyl groups of the phenantrenic ring of morphine to carrier proteins. In such context, two structural intermediate products from morphine were sucessfully synthesized by different research groups and used for the development of vaccine formulations, namely, the 3-ortho-morphine-carboxymethyl-ether product (3-O-carboxymethylmorphine, see example 2) and the morphine-6-hemisuccinate (see example 3) (S. Spector and C. W. Parker, Science, 168:1347, 1970; S. J. Spector, J. Pharmacol. Exp. Ther. 178:253, 1971; H. Van Vunakis et al., J. Pharmacol. Exp. Ther. 180:514, 1972; and S. Gross et al., Immunochemistry, 11:453-456, 1974). With regard to the aforementioned intermediate derivaties of morphine used to develop vaccine formulations, two identical patent registries published on Sep. 13, 1991 (CH 678394 A5) and May 15, 1996 (EP 0 496 839 B1) by Erich Hugo Cerny, claim invention on the structural synthesis of novel anti-morphine vaccine formulations. However, it's worth to mention, that both of these patent registries reveal no real novelty or invention regarding the synthesis of the therapeutic vaccine formulations claimed. This argument is based on that both patent registries describe the same standard synthetic procedures previously reported to generate the intermediate 3-O-carboxy-methyl-morphine derivative used to covalently conjugate the KLH-carrier protein. In both instances, they used morphine-based and the sodium beta-chloroacetate and absolute alcohol as reagents in the reaction mixture. The other synthetic intermediate derivative used to activate the covalent linkage between morphine and carrier proteins, is the morphine succinyl ester linked to the free 6-hydroxyl group of the phenantrenic ring-structure of the morphine molecule, namely, morphine-6-hemisuccinate, (see example 3) (originally reported by B. H. Wainer et al., Science, 176:1143, 1972, A. Akbarzadeh et al., Biotechnol. Appl. Biochem, 30:139-145, 1999). In same context to the aforementioned synthetic procedures used to generate the 3-O-carboxymethylmorphine derivative for synthetizing vaccine formulations, an anti-morphine vaccine patent registry released from China (CN1196955), was unjustified granted from our own perspective, to Han Ying et al., on Oct. 28, 1998. These authors claim innovation and novelty regarding the synthetic procedures and the structural formulations of vaccine preparations to different opiate drugs, besides morphine, using same standard methods to synthetize morphine-6-hemisuccinate derivative, as previously reported (see in B. H. Wainer et al., Science, 176:1143, 1972, A. Akbarzadeh et al., Biotechnol. Appl. Biochem, 30:139-145, 1999). These authors used this intermediate derivative to haptenize morphine to BSA as carrier protein.
The structural design and synthesis of different immunogenic formulations, where morphine has been haptenized to carrier proteins such as KLH and BSA, represented the basis by which authors have invariably used chemical procedures to link covalently the intermediate derivatives of morphine 3-O-carboxymethylmorphine and morphine-6-hemisuccinate to these carrier proteins (as previously outlined in the experimental studies described above, including the aforementioned patent registries), using as cross-linker the homobifunctional chemical reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The EDC reacts with the available free carboxyl groups exposed in either the 3-O-carboxymethylmorphine or morphine-6-hemisuccinate derivatives, thus forming the corresponding two O-acylurea by-products, which are chemically reactive to generate covalent amide bonds with the epsilon (ε)-amino groups in the lateral chain of lysine residues of either BSA or KLH (see example 4).
The aforementioned studies demonstrating the feasibility to generate a humoral immune response against morphine and its structural cognate semisynthetic opiate, heroin, led to a pioneer study reported nearly 30 years ago by Bonese (K. F. Bonese et al. Nature, 252:708-710, 1974). This study was in fact the pioneer report demonstrating that active vaccination with an immunogenic morphine conjugate in a single experimental animal, the non-human primate Macacus rhesus, was able to generate a protective humoral-mediated immune response that blunt the addictive self-administration behavior to heroin. The synthesis of this immunogenic conjugate was achieved by covalently haptenizing morphine to the BSA through a stable ester bond formed by the condensation of the succinic anhydride and the 6-hydroxyl group in the phenantrenic structure of the morphine molecule. The synthesized intermediate derivative, morphine-6-hemisuccinyl, was then covalently linked to the 1-ethyl-3-(3-Dimethylaminopropyl) carbodiimide (EDC) reagent, thus obtaining the complete immunogenic conjugate. The repeated subcutaneous injection of this immunogen into the primate triggered a humoral immunological response with specific morphine antibodies, which displayed cross-recognition for heroin. Additionally, the active vaccination approach with this conjugate demonstrated to be an effective procedure to blunt the re-acquisition of the intravenous self-administration behavior to heroin in this single primate, previously trained to self-administer different dose-units of this opiate. Although this pioneer report showed the first successful antibody-based antagonism procedure to blunt heroin-intake behavior in the primate, it was never patented and developed for clinical use. Similarly, no further experimental studies related to the design, synthesis and validation of further novel structural anti-morphine/heroin vaccine formulations using carrier proteins suitable for human vaccination were developed, basically due to the fact that BSA is not a licensed carrier protein for such experimental purposes. The main reason that these immunopharmacological studies were never approached in humans with suitable immunogens for morphine or heroin could be due, at leat in part, to the simultaneous and continuous development of other neuropharmacological agents used to treat morphine-heroin addiction. For instance, synthetic drugs that display weak and partial agonist activities on the mu opioid receptor (i.e., methadone and buprenorphine) and other drugs which display antagonist activities on opioid receptors (i.e., naltrexone and naloxone). All these drugs are currently used for preventing relapse to opiate addiction.
Based on the aforementioned reports of experimental vaccines against morphine/heroin addiction, which never approached human vaccination, preliminary experimental studies conducted by our research group served as a basis, for the development of the present invention of the bivalent vaccine formulation against morphine-heroin addiction. These experiments describe the design, synthesis and evaluation of the immunogenicity induced by different synthetized structural models of a new generation of vaccines against anti-morphine/heroin (B. Anton and P. Leff., 31st Annual meeting of the Society for Neuroscience. San Diego, Calif. Nov. 10-16, 2001). Such structural formulations of vaccines were intially synthetized by linking covalently the morphine-6-hemisuccinate (M-6-H) intermediate derivative with several carrier proteins such as BSA, KLH and the recombinant cholera toxin-β-subunit protein. These coupling reactions used standard crosslinking procedures for linking the morphine-6-hemisuccinate (M-6-H) intermediate derivative to the 1-ethyl-3-(3-Dimethylaminopropyl)-carbodiimide (EDC) reagent. These preliminary experimental data gathered from such studies made possible the identification of candidate carrier proteins for covalent haptenization of morphine. It is worth to mention that this work was only presented in a slide session at the aforementioned International Neuroscience Meeting.
Furthermore, it did not show any information concerning experimental data related to the design of the structural molecular models of immunogens, methodologies describing the synthesis, purification, application and dosification procedures of these new vaccines. Moreover, no references or descriptions of the validation of the anti-addictive effects against morphine-heroin were also made, which are disclosed in the present invention of the therapeutic bivalent morphine-heroin vaccine formulation against the addiction to these opiate substances.
In addition to the pioneer study reported by Bonese and co-workers, who demonstrated the efficacy of the active vaccination with BSA-morphine to blunt the addictive self-taking behavior of heroin in a single primate, other research groups explored the immunoprotective effects of the passive immunization against morphine, using behavioral paradigms of intravenous self-administration of heroin in rodents (P. R. Pentel et al., Pharmacol. Biochem. and Behavior, 9:347-352, 1991). From a potential therapeutical viewpoint, a passive immunoprotection procedure against morphine and heroin addiction has practical limitations to prolong and maintain abstinence to opiate drugs in humans on a long-term basis, as opposed to the active immunization procedures. These limitations are based on some practical observations derived from experimental results of passive immunoprotection paradigms (R. A. Goldsby, Vaccines. In: Kuby Immunology, 4th ed. Freeman and Co, New Cork, N.Y., pp. 449-466, 2001). These data have demonstrated the relatively short biological half-life of murine monoclonal antibodies (3-23 days, depending on the immunoglobulin class and isotype) after being administered in vivo into different experimental animals. Thus, immunoprotection conferred via passive administration of murine monoclonal antibodies into non-murine hosts is usually short-lived. Moreover, as both monoclonal and polyclonal antibodies used in passive immunization therapies are commonly produced from different animal species (e.g., mouse, rabbit, etc.), such immunoglobulins are usually recognized as foreign antigenic molecules when injected into human subjects. In this context, passive immunization of patients with such immunoglobulins would trigger a rapid humoral immunological response against these molecules, which ultimately result in a blunted antibody-mediated neutralizing responses and reduced half-life of these types of immunoglobulins in plasma. Thus, once primed such antigenic responses against heterologous antibodies in passively immunized subjects, the subsequent administration of these types of immunoglobulins would lead to the development of abnormal immunological responses of hypersensibility upon repeated passive administration of such molecules.
Aims and Advantages of the Invention
Based on the aforementioned background regarding the anti-addictive-based therapies against opiate abuse, specifically against morphine and heroin addiction, we can conclude that no efficacious and atoxic drugs are yet available in humans for treating and maintain prolong abstinence and relapse prevention from addictive opiate-intake behaviors. Thus far, reasons exist to justify the current need for the development, application and validation of combined new drugs and therapeutic strategies to maintain prolonged abstinence with efficacy for preventing relapse to addictive drug-intake behaviors to highly addictive opiate drugs such as heroin and morphine in the humans.
As previously mentioned, the previous experimental studies reporting the pre-clinical evaluation of different immunological strategies against drug addiction in animal models, support the potential therapeutic approach of the different immunoprotective strategies against cocaine, nicotine and opiate addiction (specifically to both morphine and heroin). This strategies include new pharmacological treatments based on antibody antagonism for the maintenance of prolong abstinence and/or prevention of relapse to drug intake and drug addiction to the aforementioned substances in the humans. In fact, the most important legacy of these immunoprotective studies against drug addiction, is the identification of the critical experimental achievements from both pre-clinical and clinical Phase studies, which ultimately would lead to the potential use and validation of these immunological strategies in humans to maintain prolong abstinence and/or to relapse prevention during addictive disorders to illegal drugs of abuse.
In this context, we can mentioned the following experimental requirements to be meet: a) design and synthesis of structural formulations of anti-addictive vaccines where the haptenic drug is structurally coupled via very stable covalent links (i.e., amide), using bifunctional chemical compounds with low structural complexity and immunogenicity that enhance the covalent crosslinking of the haptenic drug with licensed carrier proteins used in vaccination protocols in the humans; b) such proteins should display proven atoxicity, and should be able to confer a very high immunogenicity to the haptenic drug when the drug-protein conjugate is administered in active immunization protocols; c) systematic evaluation of the humoral immune response to the haptenic drug conjugated to the carrier protein, so that functional parameters of triggered specific antibodies such its titers, specificity and avidity can be properly characterized after application of the immunoconjugate in ad hoc active vaccination protocols; d) systematic monitoring of the humoral immune response during the active vaccination protocls with the immunogenic conjugate containing the haptenized addictive drug, so as to identify and assess the establishment of a long-term and stable humoral memory response against the antigenic drug after completion of the active immunization protocols; e) assessing of the capacity and efficacy of these new therapeutic vaccine formulations against drug addictions (i.e., morphine-heroin) with proven capacities to confer long-term immunoprotection against addictive drugs. These vaccines should exhibit a good therapeutic index to prevent the reacquisition of the addictive behaviors in detoxified and abstinent subjects.
The present invention relates to the design, synthesis, purification, application and validation of a novel structural model of vaccine against both morphine and heroin addiction, that fulfills all the aforementioned structural and functional requirements. The detailed description of the synthesis procedures and the structure of this carrier protein-morphine conjugate, are disclosed in the present invention. Other information also disclosed therein are its use in paradigms of active immunization in the rodent, the monitoring and characterization of the development of the humoral immune response after boosting, as well as the titers and the antibody specificity generated against the haptenized drug. Additionally, expefrimental data are also disclosed showing the proven efficacy of the present invention of the therapeutic formulation of a novel bivalent vaccine against morphine/heroin to induce a robust humoral immune response of high and sustained serum antibody titers against morphine, with equivalent specificity for heroin, in detoxified and abstinent subjects addicted to these opiate substances. Such antibodies can efficiently antagonize (block) the reacquisition of the addictive drug-intake and drug-seeking behaviors, in addition to prolong the abstinence state against these two opiate substances in hyperimmune subjects challenged to drug's reacquisition in a standard addictive intravenous self-administration paradigm of these two opiate substances in the rat.
The first aim of the present invention is to disclose the method of synthesis and the structural formulation of a novel morphine immunogen, which has the following functional and structural advantages never presented in any other previously synthetized morphine/heroin vaccine formulations: a) covalent morphine haptenization to the tetanus toxoid, a carrier protein licensed in active vaccination protocols in the human with proven capacity to confer a very high immunogenicity to hapten molecules of low molecular mass; b) covalent morphine haptenization to the tetanus toxoid, through the sequential use and covalent linkage of two different crosslinking reagents, which enhances the synthesis of a long and low immunogenic spacer linker-arm placed between the carrier protein and the haptenized drug; c) this novel morphine immunogen was dosified in active vaccination protocols in subjects, and showed a proven efficacy to generate a robust and sustained humoral immune response characterized by highly specific serum antibodies with equivalent specificities against morphine and its structurally related and highly addictive opiate analogue, heroin.
Another aim of the present invention is to disclose and validate an active immunization paradigm using the aformentioned carrier protein-morphine conjugate, for optimizing a robust and sustained humoral immune response with very high anti-hapten antibody titers with an established long-term immune memory response.
Another aim of the present invention deals with the demonstration for optimizating the antibody titer and specificity, in order to demonstrate its functional capacity for cross-recognizing heroin, but not other opiate medications structurally-related to morphine and/or several endogenous opioid peptides produced in the brain.
Another aim of the present invention concerns with the validation on the use of this immunogen, as a novel pharmaceutical composition or therapeutical formulation to confer immunoprotection against the re-acquisition of addictive morphineheroin-intake behaviors and for the maintenance of prolonged abstinence in experimental subjects previously detoxified from either morphine or heroin addiction.
An additional aim of the present invention discloses the synthesis and the molecular structure of a therapeutic anti-morphine/heroin vaccine formulation, validated in pre-clinic studies in the rodent, where this vaccine formulation containing the tetanus toxoid used as the immunogenic carrier protein to covalently haptenized morphine, provides its potential use to evaluate its therapeutical effects in clinical phases studies, by confering a long-term immunoprotection, maintenance of prolonged abstinence and relapse prevention in detoxified subjects from either morphine or heroin addiction.
A final aim of the present invention discloses the general methodology used to design, develop, apply and validate an efficient and atoxic therapy, whose mechanism of action differed from the available current classical therapeutic compounds, by enhancing pharmacokinetic changes of the aforementioned opiate drugs, thereby reducing efficiently their blood-brain barrier permeation, once they have been administered to a hyperimmune subjects, previously vaccinated against these two opiate drugs of abuse.
Collectively, the present invention discloses and provides a full description of the methodology and processes required to prepare intermediate derivatives for the synthesis of a morphine/heroin vaccine, pharmaceutical compositions or therapeutic formulations, including methods and therapeutical uses against morphine-heroin addiction.